The present invention relates generally to the art of separating charged molecular species, and, in particular, to new compositions and methods for practicing same.
Capillary gel electrophoresis is one of the most widely used separation techniques in the biologically related sciences. Charged molecular species such as proteins, peptides, nucleic acids, amino acids and oligonucleotides are separated by causing the species to migrate in a buffer medium under the influence of an electric field. The buffer medium normally is used in conjunction with a low to moderate concentration of an appropriate gelling agent, such as for example, agarose or cross-linked polyacrylamide, to promote the separation and to minimize the occurrence of mixing of the species being separated.
Until recently, electrophoretic separations were conducted in gel slabs or open gel beds which were typically fabricated of agarose or cross-linked polyacrylamide material. More recently, capillary gel electrophoresis techniques combined with photometric detection methods have allowed the automation and rapid quantitative analysis of charged molecules. Furthermore, capillary gel electrophoresis can provide quantitative information about a sample using very small amounts of the sample, gel and buffer relative to traditional slab gel processes. Moreover, high resolution separation of molecules having different effective charges have been achieved by applying electrophoretic principles to polymer solution filled or gel filled narrow capillary tubes.
Typically, the capillary columns used in capillary gel electrophoresis are fabricated from lengths of fused silica tubing having diameters on the order of 25 .mu.m to 200 .mu.m and lengths from about 30 cm to about 200 cm. The buffer and gel separation media are pumped directly into the column interiors and electrophoretic techniques are used to separate charged molecular species.
A major drawback to the use of such capillary gel electrophoresis methods, however, is the inability of many separation media to bind directly to the inner wall of capillary tubes. The inability of a separation medium to bind to the inner wall of a fused silica capillary tube which is negatively charged creates an electro-osmotic flow when an electric field is applied during electrophoresis. Such migration does not provide a satisfactory separation of the constituent parts of a sample.
Accordingly, traditional methods aimed at preventing electro-osmosis include introducing a compound which binds to the inner surface of a capillary tube wall, as well as, to the separation medium prior to injecting the separation medium into the tube. For example, U.S. Pat. No. 5,447,617 to Shieh describes covalently bonding polybutadiene to the inner surface of a capillary tube, introducing polyacrylamide therein and co-polymerizing the polyacrylamide with the polybutadiene. Such precoating techniques, however, are time consuming, inconvenient and costly.
A further problem of conventional capillary gel electrophoresis is encountered with the use of polyacrylamide-based separation media. Such media are injected into the capillary tube in unpolymerized form. Polymerization of the polyacrylamide is then induced within the capillary tube by any number of methods including ultraviolet radiation and chemical catalysts. Such methods are characterized by a lack of uniformity in the pore size distribution of the polymer network formed, and by incomplete polymerization.
A still further problem encountered with the use of polyacrylamide-based separation media in capillary gel electrophoresis methods relates to the irreversible nature of the polymerized polyacrylamide gel. Once the polyacrylamide is polymerized within a capillary electrophoresis tube, the polymerized gel is very difficult to remove from the capillary tube after electrophoresis. Furthermore, isolation of the separated constituents of a sample from the polymerized polyacrylamide gel is very difficult because of the polymerized gel.
Accordingly, attempts have been made to use nonpolymerized separation media for capillary gel electrophoresis. For example, U.S. Pat. No. 5,126,021 to Grossman describes a capillary electrophoresis (CE) element composed of an uncharged, water-soluble polymer in a low-viscosity solution. The polymers within this solution form entanglements which create a mesh size that is suitable for electrophoretic separation of charged molecules. This CE element remains liquid during the electrophoretic separation of a sample which allows for easy removal of the element from the capillary tube. The Grossman '021 patent, however, requires that a coating be deposited onto the inner surface of the capillary tube prior to introduction of the electrophoresis element in order to prevent electro-osmotic flow. As set forth hereinabove, such a precoating is time consuming, inconvenient and costly.
Similarly, U.S. Pat. No. 5,468,365 to Menchen et al. describes an electrophoresis medium having a matrix of aggregated copolymers dispersed in an aqueous medium. The polymer matrix of the '365 patent is described as a dispersion of one substance (micelles) in another (water). In such a dispersion, the particles are formed by the association or aggregation of molecules having both hydrophilic and hydrophobic regions. The copolymers of the '365 patent form a polymer matrix having a relatively uniform mesh size which is believed to be related to the regular, i.e., substantially uniform spacing between adjacent hydrophobic polymer segments. The separation matrix of the '365 patent, however, exists only in one phase (liquid) and is accordingly difficult to manipulate.
Accordingly, it would be desirable to provide a new separation medium for electrophoresis methods that does not require coating of the inner cavity of a capillary tube. It would also be desirable to provide a new separation medium with the ability to change between liquid and gel-like states for efficient application and removal of the medium from electrophoresis apparatus. In particular, it would be desirable to provide a viscosity-adjustable medium for use in separation methods which is easy to apply and remove from various apparatus, and from which it is easy to isolate components of an electrophoretically separated sample.